It is of great significance for electrochemical sensors to simultaneously detect dopamine (DA) and uric acid (UA) related to biological metabolism. In this work, two-dimensional (2D) porous carbon nanosheets (CNS) was prepared as electrocatalysts to improve the sensitivity, the selectivity, and the detection limit of the simultaneous detection. First, 2D amorphous iron-metal organic frameworks (Fe-MOF) was synthesized with Fe3+ and terephthalic acid via a facile wet chemistry method at room temperature. And then, CNS was prepared by pyrolysis and pickling of Fe-MOF. CNS had large specific surface area, good electrical conductivity and lots of carbon defects. The response currents of the CNS modified electrode was larger than those of the control electrodes in the simultaneous determination. The simultaneous determination were measured via difference pulse voltammetry to reduce the effect of capacitive currents on quantitative analysis. The CNS modified electrodes showed high sensitivity and low detection limit for the simultaneous detection of DA and UA. The modified electrodes have been successfully used to detect DA and UA in normal human serum.